Response to Qian and Colvin: Zinc-mediated Regulation of the Cardiac Ryanodine Receptor Occurs via Multiple Binding Sites [Letters to the Editor]

This is a response to a letter by Qian and Colvin (1).We would like to thank Qian and Colvin for their interest in our recent publication (2) where we demonstrate for the first time that Zn2+ acts as a high affinity activator of the cardiac ryanodine receptor (RyR2). We are aware that BAPTA (2,2′-(ethylenedioxy)dianiline-N,N,N′,N′-tetraacetic acid) is not a Ca2+-specific chelator and can also bind Zn2+ when present. The purpose of the experiment represented in Fig. 4 was to show that Zn2+ can directly activate RyR2 when levels of Ca2+ are subactivating rather than to provide the absolute Zn2+ concentration required for Zn2+-dependent channel openings. This was addressed in the experiments carried out in the absence of BAPTA, where our data reveal that Zn2+ is the primary activating ligand of RyR2 at concentrations >1 nm (Figs. 1 and 3). The estimates of free Zn2+ levels in the presence of BAPTA offered by Qian and Colvin in no way alter the interpretation of our data, and it is unclear why this led them to speculate that the action of Zn2+ is through a single site on the channel (1). A single-site model is not consistent with the finding that 100 pm Zn2+ sensitizes Ca2+-mediated RyR2 activity yet higher concentrations of Zn2+ (1–100 nm) enable switching from Ca2+-dependent to Ca2+-independent gating. Thus, separate Zn2+ sites must exist to enable Ca2+ sensitization and Zn2+ activation, respectively. A single-site model is also not consistent with the observation that very high concentrations of Zn2+ (1 mm)...

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